Comparison of genomic profiling of circulating tumor DNA in pancreaticobiliary malignancies in plasma and bile.

BACKGROUND: Obtaining sufficient pancreaticobiliary tumor tissue for genomic profiling has limitations. Liquid biopsies using plasma do not provide sufficient sensitivity. Thus, this study aimed to determine the effectiveness of liquid biopsy between bile and plasma for identifying oncogenic and drug-matched mutations. METHODS: This study created a panel of 60 significantly mutated genes specific to pancreaticobiliary cancer (PBCA) and used it for genomic analysis of 212 deoxyribonucleic acid (DNA) samples (87 bile supernatant, 87 bile precipitate, and 38 plasma) from 87 patients with PBCA. The quantity of extracted DNA from bile and plasma was compared, as were genomic profiles of 38 pairs of bile and plasma from 38 patients with PBCA. Finally, we investigated 87 bile and 38 plasma for the ability to detect druggable mutations. RESULTS: The amount of DNA was significantly lower in plasma than in bile (p < .001). Oncogenic mutations were identified in 21 of 38 (55%) patients in bile and nine (24%) in plasma samples (p = .005). Bile was significantly more sensitive than plasma in identifying druggable mutations (p = .032). The authors detected 23 drug-matched mutations in combined bile and plasma, including five ERBB2, four ATM, three BRAF, three BRCA2, three NF1, two PIK3CA, one BRCA1, one IDH1, and one PALB2. CONCLUSIONS: Liquid biopsy using bile may be useful in searching for therapeutic agents, and using the obtained genomic information may improve the prognoses of patients with PBCA. PLAIN LANGUAGE SUMMARY: Genomic profiling of formalin-fixed paraffin-embedded tissues may provide actionable targets for molecular and immuno-oncological treatment. However, most pancreaticobiliary malignancies are unresectable and formalin-fixed paraffin-embedded tissues cannot be obtained. Although comprehensive genomic profiling tests using plasma have been used in recent years, the utility of those using bile is not clear. Our study revealed that bile identified more drug-matched mutations than plasma in advanced pancreaticobiliary cancer patients. Bile may help widen the patient population benefiting from targeted drugs.

Carcinogenic potential in regenerated mucosa after endoscopic resection of esophageal squamous cell carcinoma.

BACKGROUND AND AIM: Little is known about genetic mutations in the regenerated mucosa (RM) after endoscopic resection (ER) of esophageal carcinoma. Thus, this study investigates the status of genetic variation in RM after ER of esophageal squamous cell carcinoma (ESCC). METHODS: The study cohort included 19 patients with ESCC. We used an esophageal carcinoma panel to identify target sequences for squamous cell carcinoma (SCC), background mucosa (BM), and RM after ER of ESCC. We used OncoKB to check whether each mutation was a putative driver. RESULTS: We identified 77 mutations of 32 genes in SCC, 133 mutations of 34 genes in BM, and 100 mutations of 29 genes in RM. Putative driver mutations were identified in 20 mutations in 14 cases in SCC, 16 mutations in 10 cases in BM, and 7 mutations in 11 cases in RM. The rate of putative driver mutations to total mutations was significantly lower in RM (26% in SCC vs 12% in BM vs 7% in RM, P = 0.009). Additionally, the rate of cases with TP53 putative driver mutations was significantly lower in RM (63% in SCC vs 37% in BM vs 16% in RM, P = 0.011). The percentage of putative driver mutations and the percentage of cases with a putative driver of TP53 were significantly lower in RM. CONCLUSION: Esophageal RM after ER of ESCC could have a lower risk of carcinogenesis.

Influence of formalin fixation duration on RNA quality and quantity from formalin-fixed paraffin-embedded hepatocellular carcinoma tissues.

Analyzing RNA samples from formalin-fixed paraffin-embedded (FFPE) tissues is essential for precision medicine. We investigated RNA quantity and quality from FFPE tumor tissues fixed in formalin for various times and compared sequencing metrics from next-generation sequencing (NGS). Hepatocellular carcinoma (HCC) tissues were fixed in 10% neutral buffered formalin (1-240 h) and FFPE blocks were prepared. Total RNA was extracted, and the quantity and quality were assessed using the NanoDrop, Qubit and Bioanalyzer. After preparing sequencing libraries, NGS was performed on the Oncomine Dx Multi-CDx system. Total RNA yields of all samples met the threshold required for NGS, but longer fixation times resulted in decreased total RNA and long RNA fragment (>200 nt) yields. NGS analysis showed fewer sequencing reads of internal control genes from RNA with longer fixation times. RNA extracted from FFPE blocks stored for 500 days had reduced RNA yield and quality compared with RNA obtained from FFPE blocks prepared immediately. In conclusion, short and over-fixation should be avoided because of their negative impact on sequencing quality. Fixation process should be finished promptly within recommended guidelines (6-72 h) for cancer patients.

PIK3CA-AKT pathway predominantly acts in developing ipsilateral breast tumor recurrence long after breast-conserving surgery.

PURPOSE: Ipsilateral breast tumor recurrence (IBTR) after breast-conserving therapy is seen after a long interval, but the clinical classification of Residual Tumor Recurrence (RR) or Double Primary (DP) needs to be validated. We used genome profiling to identify the genetic alterations associated with IBTR. METHODS: Among 1881 breast cancer patients treated with breast-conserving therapy between 1999 and 2018, IBTR occurred in 52 patients (2.8%). Of these 22 patients who consented for genomic analysis of Primary Breast Cancer (T1) and IBTR (T2) were studied. When the same gene mutations in T1 and T2 were identified, it was classified as genomic residual recurrence gRR, and when no shared mutations identified, it was classified as gDP. The differences between clinical and genomic classification were compared. Furthermore, the pathway of the genes which were responsible for recurrence was also examined. RESULTS: Of 13 clinically diagnosed RRs (cRRs), 11 were gRR and 2 were gDPs, while of 9 cDPs, 6 were gDP and 3 gRR, with a match rate of 17/22 (77%). We searched for genes involved in IBTR: PIK3CA-AKT pathway mutations were found in 12 of 14 gRRs (86%) in T1, and only 2 of 8 gDPs (25%) with significant difference (p = 0.004). When both of PBC and IBTR compared, PIK3CA-AKT pathway abnormalities were 24/28 (86%) in the gRR and 5/16 (31%) in the gDP (p < 0.001). CONCLUSIONS: Genome profiling revealed that abnormalities in the PIK3CA-AKT pathway in long-term residential recurrences and are a crucial molecular group in the development of IBTR.

TP53-positive clones are responsible for drug-tolerant persister and recurrence of HER2-positive breast cancer.

PURPOSE: The prognosis of HER2-positive breast cancer has improved with the development of anti-HER2 therapies. In order to further improve the prognosis of HER2-positive breast cancer, it is essential to elucidate the cells that survive during the therapy (drug-tolerant persister DTP). METHODS: Of the 2022 breast cancer patients operated at our institution during 2004-2018, 240 (12%) had HER2-positive breast cancer. Neo-adjuvant chemotherapy including trastuzumab (Tr-NAC) was administered to 94 of them. Forty-six of them were complete remission (CR), and 48 were non-CR. After 6.9 ± 3.7 years of follow-up, all 46 CR cases showed no recurrence (Cohort A), and 48 non-CR cases were divided into 31 cases with no recurrence (Cohort B) and 17 cases with recurrence (Cohort C). In addition to clinical backgrounds, we compared genomic profiles for 27 patients (Cohort A; 15/48, B; 7/31, and C; 5/17) who consented to genomic analysis. RESULTS: Genomic abnormalities of TP53 and PIK3CA were frequently observed in biopsy samples pre Tr-NAC, but we found no differences between CR (Cohort A) and non-CR (Cohorts B + C). Then, we examined both of pre and post Tr-NAC samples of Cohort B (7) and C (5) to see the relationship between recurrence and genomic abnormalities. TP53 mutations were significantly more prevalent in Cohort C (5/5, 100%) than cohort B (3/7, 43%) in the surgical sample after treatment (p = 0.04). PyClone analysis of TP53 mutations showed that the cellular frequency of TP53 clones increased in 4 of 5 patients in Cohort C and none of B. On the other hand, we found no enhancement of PIK3CA mutant clones in Cohort C. CONCLUSIONS: The DTP after Tr-NAC associated with subsequent relapse had TP53 mutations, suggesting that overcoming DTP with TP53 mutations is the most important clinical challenge. TRIAL REGISTRATION: Not applicable.

Direct comparison of Xpert Xpress, FilmArray Respiratory Panel, Lumipulse antigen test, and RT-qPCR in 165 nasopharyngeal swabs.

BACKGROUND: The nucleic acid amplification test (NAAT) and antigen test are approved diagnostic tests for COVID-19. In this study, we aimed to investigate the assay performance of two NAATs (Xpert Xpress SARS-CoV-2 and FilmArray Respiratory Panel) and a quantitative antigen test (Lumipulse). METHODS: One hundred and sixty-five nasopharyngeal swabs were subjected to Xpert, FilmArray, Lumipulse, and RT-qPCR assays. RESULTS: Of 165 samples, RT-qPCR showed 100 positives and 65 negatives. The Xpert had an overall agreement of 99.4% (95% confidence interval [CI]: 96.7-99.4%), sensitivity of 99% (95% CI: 96.8-99%), and specificity of 100% (95% CI: 96.6-100%). FilmArray had an overall agreement of 98.8% (95% CI: 95.9-98.8%), sensitivity of 98% (95% CI: 95.6-98%), and specificity of 100% (95% CI: 96.3-100%). Lumipulse had an overall agreement of 95.5% (95% CI: 91.8-95.5%), sensitivity of 92.3% (95% CI: 89.2-92.3%), and specificity of 100% (95% CI: 95.5-100%). The κ coefficient showed excellent agreement between each test and RT-qPCR. There was a high correlation between Xpert Ct values, RT-qPCR Ct values, viral loads and antigen level. CONCLUSIONS: Xpert Xpress and FilmArray Respiratory Panel exhibited an equivalent performance. The Lumipulse antigen test was slightly less sensitive than the NAATs, but showed high assay performance except for samples with low viral load. The Xpert Xpress, FilmArray Respiratory Panel and Lumipulse antigen tests offer rapid sample-to-answer data, allowing random access detection on automated devices.

A case of juvenile-onset pheochromocytoma with KIF1B p.V1529M germline mutation.

In 2008, a familial noradrenergic pheochromocytoma (PCC) with a KIF1B germline mutation in exon 41 was reported in a 24-year-old female proband and her family. However, in 2020, the same research group reported that the cause of PCC in this family was a MAX germline mutation and was not due to the KIF1B mutation. In this study, we investigated the pathogenicity of a KIF1B germline mutation detected in a 26-year-old woman with juvenile-onset noradrenergic PCC. She was surgically treated and did not have a family history of PCC. We performed whole-exome sequencing, Sanger sequencing, and immunohistochemical and gene expression analyses of catecholamine-synthesizing enzymes. Three tumors with associated somatic mutations were used as the control group. Whole-exome sequencing revealed a p.V1529M KIF1B germline mutation in exon 41 in our patient, and no other associated germline and somatic mutations, including MAX, were detected. Sanger sequencing confirmed the presence of both mutant and wild-type alleles in the tumor. Among the catecholamine-synthesizing enzymes, the expression of phenylethanolamine-N-methyl transferase was suppressed. An in silico analysis of the p.V1529M mutation showed a score suggestive of pathogenicity. After evaluation with the international guideline for sequence variants, p.V1529M mutation was still classified as a variant with uncertain significance; however, our data, including the in silico analysis data, provided certain evidences that met the criteria supporting its pathogenicity. Therefore, this study can support future studies in proving the pathogenicity of the KIF1B p.V1529M mutation.

Genomic profiling amplifies the utility of endoscopic ultrasound-guided fine needle biopsy by identifying clinically applicable druggable mutations in pancreatic cancer.

BACKGROUND: Genomic profiling of tumors is available, but whether the small fragment obtained via endoscopic ultrasound-guided fine needle biopsy (EUS-FNB) is sufficient for these examinations is unknown. Here we investigated whether EUS-FNB specimens are suitable for genomic profiling to identify oncogenic and drug-matched mutations. METHODS: We constructed a pancreatobiliary cancer panel for targeted panel sequencing that covered 60 significantly mutated genes and compared the results with those of whole-exome sequencing (WES). In total, 20 and 53 formalin-fixed paraffin-embedded tissues obtained via surgery and EUS-FNB were analyzed, respectively. First, we examined the DNA quality and genomic profiles of 20 paired samples from 20 malignant lesions obtained via surgery and EUS-FNB. We then tested 33 samples obtained via EUS-FNB from 24 malignant and 9 benign lesions for the discrimination of malignancy. Finally, we explored drug-matched mutations from EUS-FNB specimens. RESULTS: Although the DNA quantity obtained via surgery was higher than that obtained via EUS-FNB (P = 0.017), the DNA quality and mean depth were equivalent (P = 0.441 and P = 0.251). Panel sequencing of EUS-FNB specimens identified more oncogenic mutations than WES (90 % vs. 50 %). Furthermore, the number of oncogenic mutations did not differ between EUS-FNB and surgically resected specimens. Genomic profiling of EUS-FNB specimens enabled the discrimination of malignancy with 98 % accuracy. Of 44 malignant lesions, drug-matched alterations were identified in 14 % (6/44) of malignant lesions. CONCLUSION: EUS-FNB specimens can be widely utilized for diagnostic purposes, discrimination of malignancy, and detection of drug-matched mutations for the treatment of pancreatic cancer.

Detection of actionable mutations in cytological specimens obtained by endoscopic ultrasound-guided fine needle aspiration with rapid onsite evaluation in pancreatic cancer.

BACKGROUND: It is not clear whether archived cytological specimens (ACSs) obtained with endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) with rapid onsite evaluation (ROSE) can be used for genomic profiling of tumors. We used ACSs to perform genomic analysis of specimens to identify oncogenic and druggable mutations. METHODS: A panel of 60 significantly mutated genes specific to pancreatobiliary cancer was created and used for genomic analysis of 113 specimens of 44 formalin-fixed paraffin-embedded (FFPE) tissues and 69 ACSs obtained by EUS-FNA with ROSE were included. The quantity and quality of DNA extracted from FFPE tissues and ACSs were compared. We also compared DNA from spray and touch ACSs. Next, genomic profiles were compared. We also evaluated detection of target gene mutations in each specimen. RESULTS: The amount of DNA in FFPE tissues was greater than in ACSs (P = 0.014), but the quality of DNA was comparable (P = 0.378). There was no quantitative or qualitative difference between spray and touch ACSs (P = 0.154 and P = 0.734, respectively). Oncogenic mutations were shared at 82 % in FFPE tissues and ACSs and 82 % in spray and touch ACSs. The sensitivity of genomic analysis in ACSs was 97 % (67 of 69), which was comparable to that of cytology (62 of 69, 90 %; P = 0.165), and was significantly higher than that of histology (32/44, 73 %; P < 0.001). Drug-matched mutations were identified in five of the 44 lesions (11 %). CONCLUSION: Genomic analysis of ACSs is useful in the prognosis of pancreatic cancer because detection of driver mutations is similar to detection in FFPE tissues.

Comprehensive Analysis of Barrett's Esophagus: Focused on Carcinogenic Potential for Barrett's Cancer in Japanese Patients.

BACKGROUND/AIM: Barrett's esophagus (BE) is a precursor of esophageal adenocarcinoma (EAC). Therefore, an accurate diagnosis of BE is important for the subsequent follow-up and early detection of EAC. However, the definitions of BE have not been standardized worldwide; columnar-lined epithelium (CLE) without intestinal metaplasia (IM) and/or < 1 cm is not diagnosed as BE in most countries. This study aimed to clarify the malignant potential of CLE without IM and/or < 1 cm genetically. METHOD: A total of 96 consecutive patients (including nine patients with EAC) who had CLE were examined. Biopsies for CLE were conducted, and patients were divided into those with IM and > 1 cm (Group A) and those without IM and/or < 1 cm (Group B). Malignant potential was assessed using immunochemical staining for p53. Moreover, causative genes were examined using next-generation sequencing (NGS) on ten patients without Helicobacter pylori infection and without atrophic gastritis. RESULT: Of the 96 patients, 66 were in Group B. The proportion of carcinoma/dysplasia in Group A was significantly higher than that in Group B (26.7% in Group A and 1.5% in Group B; p < 0.01). However, one EAC patient was found in Group B. In the immunostaining study for non-EAC patients, an abnormal expression of p53 was not observed in Group A, whereas p53 loss was observed in three patients (4.6%) in Group B. In the NGS study, a TP53 mutation was found in Group B. CONCLUSION: CLE without IM and/or < 1 cm has malignant potential. This result suggests that patients with CLE as well as BE need follow-up.

Analysis of a persistent viral shedding patient infected with SARS-CoV-2 by RT-qPCR, FilmArray Respiratory Panel v2.1, and antigen detection.

Various diagnostic tests utilizing different principles are currently under development for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, these tests can occasionally produce discrepant results, causing confusion in their interpretation. Here, we evaluated the performance and features of three diagnostic assays: quantitative reverse transcription polymerase chain reaction (RT-qPCR), FilmArray Respiratory Panel (RP) v2.1, and the LUMIPULSE antigen test. Twenty-seven serial nasopharyngeal swabs were collected from a prolonged viral shedding patient who had been hospitalized for 51 days. We examined the SARS-CoV-2 detection rates of the three tests. The overall agreement rate was 81% between RT-qPCR and FilmArray RP v2.1, 63% between the antigen test and FilmArray RP v2.1, and 59% between the antigen test and RT-qPCR. We obtained concordant results in samples with high viral loads (low threshold cycle values) by all three tests. RT-qPCR and FilmArray RP v2.1 accurately detected SARS-CoV-2 at the early to intermediate phases of infection, but the results varied at the late phase. The antigen test also produced a positive result at the early phase but varied at the intermediate phase and consistently produced negative results at late phase of infection. These results demonstrated FilmArray RP v2.1 could detect SARS-CoV-2 with accuracy comparable to RT-qPCR. Further, there were discrepant results using different types of diagnostic tests during the clinical course of prolonged viral shedding patient. We provided insights into how to utilize different types of kits to assess and manage SARS-CoV-2 infections.

Genomic profile of urine has high diagnostic sensitivity compared to cytology in non-invasive urothelial bladder cancer.

Cytology is widely conducted for diagnosis of urothelial bladder cancer; however, its sensitivity is still low. Recent studies show that liquid biopsies can reflect tumor genomic profiles. We aim to investigate whether plasma or urine is more suitable for detecting tumor-derived DNA in patients with early-stage urothelial bladder cancer. Targeted sequencing of 71 genes was carried out using a total of 150 samples including primary tumor, urine supernatant, urine precipitation, plasma and buffy coat from 25 patients with bladder cancer and five patients with cystitis and benign tumor. We compared mutation profiles between each sample, identified tumor-identical mutations and compared tumor diagnostic sensitivities between urine and conventional cytology. We identified a total of 168 somatic mutations in primary tumor. In liquid biopsies, tumor-identical mutations were found at 53% (89/168) in urine supernatant, 48% (81/168) in urine precipitation and 2% (3/168) in plasma. The high variant allele fraction of urine was significantly related to worse clinical indicators such as tumor invasion and cytological examination. Although conventional cytology detected tumor cells in only 22% of non-invasive tumor, tumor diagnostic sensitivity increased to 67% and 78% using urine supernatant and precipitation, respectively. Urine is an ideal liquid biopsy for detecting tumor-derived DNA and more precisely reflects tumor mutational profiles than plasma. Genomic analysis of urine is clinically useful for diagnosis of superficial bladder cancer at early stage.

Rapidly declining trend of signet ring cell cancer of the stomach may parallel the infection rate of Helicobacter pylori.

BACKGROUND: Studies indicate that gastric cancer (GC) incidence has decreased, whereas signet ring cell carcinoma (SRC) incidence has increased. However, recent trends in GC incidence are unclear. We used our hospital cancer registry to evaluate the changes in the incidence of GC, SRC, and non-SRC (NSRC) over time in comparison to changes in the H. pylori infection rates over time. METHODS: We identified 2532 patients with GC enrolled in our registry between January 2007 and December 2018 and statistically analyzed SRC and NSRC incidence. The H. pylori infection rate in patients with SRC was determined by serum anti-H. pylori antibody testing, urea breath test, biopsy specimen culture, and immunohistochemical analysis (IHC) of gastric tissue. Additionally, genomic detection of H. pylori was performed in SRCs by extracting DNA from formalin-fixed paraffin-embedded gastric tissue and targeting 16S ribosomal RNA of H. pylori. RESULTS: Overall, 211 patients had SRC (8.3%). Compared with patients with NSRC, those with SRC were younger (P <  0.001) and more likely to be female (P <  0.001). Time series analysis using an autoregressive integrated moving average model revealed a significant decrease in SRC (P <  0.001) incidence; NSRC incidence showed no decline. There was no difference in H. pylori infection prevalence between the SRC and NSRC groups. IHC and genomic methods detected H. pylori in 30 of 37 (81.1%) SRCs. CONCLUSIONS: Reduction in H. pylori infection prevalence may be associated with the decrease in the incidence of SRC, which was higher than that of NSRC.

Combined annotation-dependent depletion score for BRCA1/2 variants in patients with breast and/or ovarian cancer.

Utility of combined annotation-dependent depletion (CADD) score was recently reported to rank pathogenicity as C-scores ranging 1-99 for both confirmed deleterious mutation. Using C-scores for BRCA1/2 variants, we tried to constitute the classification system for variant of uncertain significance (VUS), which had been a major problem of genetic testing for hereditary breast and/or ovarian cancer. We analyzed BRCA1/2 genes for 283 patients with breast and/or ovarian cancer. The deleterious mutation and missesne mutations, minor variant, and wild type of BRCA1 and -2 were 5, 27, 251 and 15, 85, 183, respectively. Meanwhile, the variants with C-score ≥10 were involved in 19/283 (6.7%) in BRCA1 and 34/283 (12%) in BRCA2. All deleterious mutations were included in this group. Frequency of personal history and family history of ovarian cancer were significantly high, and frequency of serous adenocarcinoma of ovary and triple negative breast cancer was relatively high in the group with deleterious mutations. Similar findings were seen in patients with variants of C-score ≥10. According to the C-score and population frequency, we could define VUS for 11 patients out of 283 patients (3.9 CADD is useful to classify the variant of BRCA1/2 and selecting the patient who needs further segregation studies.

Stepwise addition of genetic changes correlated with histological change from "well-differentiated" to "sarcomatoid" phenotypes: a case report.

BACKGROUND: Sarcomatoid cancer is defined by the World Health Organization as a category of non-small cell lung cancers with sarcoma or sarcoma-like differentiation. They are characterized by poor prognosis and resistance to conventional chemotherapy. However, the mutational profile of sarcomatoid cancer remains yet to be elucidated. Sarcomatoid cancers are usually biphasic tumors composed of carcinomatous and sarcomatous components, but the evolutional development of sarcomatoid cancer is controversial. CASE PRESENTATION: We present an illustrative case of sarcomatoid cancer composed of three different histological areas. Targeted sequencing of 53 lung cancer-related genes was performed in each component and their phenotypic changes were correlated with stepwise addition of genetic changes. CONCLUSION: Sarcomatous change of carcinoma occurs in the case of sarcomatoid cancer, and phenotypic changes to sarcomatoid cancer are associated with the addition of mutation patterns and derived from poorly differentiation tumor.

Comparison between two amplicon-based sequencing panels of different scales in the detection of somatic mutations associated with gastric cancer.

BACKGROUND: Sequencing data from The Cancer Genome Atlas (TGCA), the International Cancer Genome Consortium and other research institutes have revealed the presence of genetic alterations in several tumor types, including gastric cancer. These data have been combined into a catalog of significantly mutated genes for each cancer type. However, it is unclear to what extent significantly mutated genes need to be examined for detecting genetic alterations in gastric cancer patients. Here, we constructed two custom-made sequencing panels of different scales, the Selective hotspot Panel and the Comprehensive Panel, to analyze genetic alterations in 21 resected specimens endoscopically obtained from 20 gastric cancer patients, and we assessed how many mutations were detectable using these different panels. RESULTS: A total of 21 somatic mutations were identified by the Selective hotspot Panel and 70 mutations were detected by the Comprehensive Panel. All mutations identified by the Selective hotspot Panel were detected by the Comprehensive Panel, with high concordant values of the variant allelic fraction of each mutation (correlation coefficient, R = 0.92). At least one mutation was identified in 13 patients (65 %) by the Selective hotspot Panel, whereas the Comprehensive Panel detected mutations in 19 (95 %) patients. Library preparation and sequencing costs were comparable between the two panels. CONCLUSIONS: Our results indicate the utility of comprehensive panel-based targeted sequencing in gastric cancer.

BRCA1 and BRCA2 mutations in Japanese patients with ovarian, fallopian tube, and primary peritoneal cancer.

BACKGROUND: The contribution of BRCA1 and BRCA2 to ovarian cancer in Japanese patients is still unclear. This study investigated the frequency of germline mutations in BRCA1/2 in Japanese patients with ovarian, peritoneal, or fallopian tube cancer, regardless of their family histories, which were suggestive of hereditary breast and ovarian cancer. METHODS: Ninety-five unselected women with ovarian cancer who were seen from 2013 to 2015 at Yamanashi Prefectural Central Hospital were enrolled. Analyses of BRCA1/2 gene mutations were performed with next-generation sequencing. RESULTS: Twelve of the 95 patients (12.6%), including 5 in the BRCA1 (5.3%) and 7 in the BRCA2 (7.4%), had deleterious mutations. Among the 36 cases with a family history, 6 (16.7%) were found to carry mutations in BRCA1 and BRCA2. Notably, 6 of the 59 cases (10.2%) without a family history also had BRCA1/2 germline mutations. There was no statistical difference between the 2 groups (P = .36). The presence of mutations and their clinical relevance were studied. Mutation carriers were diagnosed at advanced stages (100% of positive cases among stage III or IV cases) and had poor prognostic histological subtypes (100% of positive cases had high-grade serous adenocarcinomas). CONCLUSIONS: In this unselected Japanese population, approximately 13% of the cases with ovarian cancer appeared to be associated with an inherited risk, regardless of a family history. This finding indicates that BRCA1/2 genetic testing should be performed for all patients with ovarian cancers.

Targeted and exome sequencing identified somatic mutations in hepatocellular carcinoma.

AIM: Genetic analysis has revealed a subset of recurrently mutated genes and aberrant cellular signaling pathways in hepatocellular carcinoma. To investigate genetic alterations and dysregulated pathways in hepatocellular carcinoma, we performed targeted sequencing and exome analysis using next-generation sequencer. METHODS: We analyzed the somatic mutational profiles of 16 genes in primary hepatocellular carcinoma by targeted ultra-deep sequencing using nine pairs of specimens (tumor and peripheral blood) and whole-exome sequencing using one pair of samples. RESULTS: Overall, somatic mutations with high allele fraction were identified in tumor tissues by targeted deep sequencing. Somatic mutations with high allele fraction were observed in TP53 (3/9; 33%) and CTNNB1 (2/9; 22%) genes in five out of nine (55%) specimens. In vitro analysis showed that CTNNB1 H36P mutant protein identified in tumor samples was resistant to protein degradation and promoted cell proliferation. Exome sequencing identified SLIT3 mutation, implying that dysregulation of axon guidance genes is involved in the development of hepatocellular carcinoma. These results showed that TP53 and WNT/ß-catenin signaling pathways were commonly mutated in hepatocellular carcinoma. CONCLUSION: These results suggest that targeted sequencing and exome sequencing enable the identification of putative oncogenic driver mutations during the development of hepatocarcinoma.

Analysis of PALB2 mutations in 155 Japanese patients with breast and/or ovarian cancer.

BACKGROUND: PALB2 (Partner and Localizer of BRCA2) was identified as a moderate-risk gene in breast and pancreatic cancers. Recently, it was reported that PALB2 carriers have a high risk of developing breast cancer, with the cumulative risk of 34 % by the age of 70. PATIENTS AND METHODS: Peripheral blood samples from 155 patients at risk for hereditary breast and/or ovarian cancer were tested for BRCA1/2 and PALB2 by targeted sequencing using a next-generation sequencer. Of these 155, 146 met NCCN criteria and the remaining 9 did not. RESULTS: BRCA1/2 analysis was performed on 155 patients, for whom the results were reported previously (Hirotsu Y et al. Mol Genet Genomic Med, doi:10.1002/mgg3.157, 2015). Eleven patients were identified to have deleterious BRCA mutations (Hirotsu Y et al. Mol Genet Genomic Med, doi:10.1002/mgg3.157, 2015). However, none of the 155 patients were found to have deleterious PALB2 germline mutations. Missense mutations [variants of uncertain significance (VUS)] of PALB2 were found in 12 cases. In silico analyses by SIFT (Sorting Intolerant Form Tolerant) and PolyPhen2 (Polymorphism Phenotyping version 2) indicated that 2 of 12 VUS were deleterious and probably damaging. CONCLUSIONS: This is the first report on PALB2 mutations in Japan, revealing two missense mutations as "deleterious and probably damaging" by in silico analyses, but no PALB2 premature truncation mutations were identified. The sample size is relatively small and a larger cohort study is needed in Japan.

Clinical and molecular biomarkers predicting response to PARP inhibitors in ovarian cancer.

OBJECTIVE: To determine the useful biomarker for predicting the effects of poly-(ADP ribose)-polymerase (PARP) inhibitors in Japanese patients with ovarian cancer. METHODS: We collected clinical information and performed molecular biological analysis on 42 patients with ovarian, fallopian tube, and primary peritoneal carcinomas who received PARP inhibitors. RESULTS: Among the analyzed patients with ovarian cancer, 23.8% had germline BRCA mutation (gBRCAm), 42.9% had homologous recombination repair-related gene mutation (HRRm), and 61.1% had a genomic instability score (GIS) of ≥42. Patients with HRRm had a significantly longer progression-free survival (PFS) than those without HRRm (median PFS 35.6 vs. 7.9 months; p=0.009), with a particularly marked increase in PFS in patients with gBRCAm (median PFS 42.3 months). Similarly, among patients with recurrent ovarian cancer, those with HRRm had a longer PFS than those without HRRm (median PFS 42.3 vs. 7.7 months; p=0.040). Multivariate Cox proportional hazards regression analysis found that performance status and gBRCAm status were independent factors associated with prolonged PFS with PARP inhibitors. In recurrent ovarian cancer, multivariate regression analysis identified platinum-free interval (PFI) in addition to performance status as a significant predictor of PFS. On the contrary, no significant association was observed between PFS and a GIS of ≥42 used in clinical practice. CONCLUSION: We found that HRRm can be a useful biomarker for predicting the effects of PARP inhibitors in treating ovarian cancer and that the PFI can also be useful in recurrent ovarian cancer.